Clostridium difficile is a gram-positive, spore-forming germ, growing strictly anaerobically, which was only identified at the end of the 1970s as an etiological agent of antibiotic-associated diarrhoea and pseudomembranous colitis. Since the 1990s, C. difficile has been regarded as the most significant hospital germ in developed countries. As a consequence of the continuously expanding use of broad spectrum antibiotics, the incidence of C. difficile infections is constantly increasing further especially in people treated as in-patients.
The exotoxins toxin A (TcdA) and toxin B (TcdB) produced by C. difficile are responsible for the C. difficile-associated diseases. Various strains exist with different virulence and toxin production. Approximately one quarter of all strains produces no toxins. Toxin-forming strains produce almost always both toxins. TcdA is an enterotoxin which through cytotoxic damage to the enterocytes increases the permeability of the intestinal mucosa and hence initiates diarrhoea. TcdB is a cytotoxin which disturbs the electrolyte transportation and is responsible for loss of fluid and functional disturbances of the intestine. The toxins TcdA and TcdB belong to the group of so-called large clostridial cytoxins (LCTs) and consist respectively of a peptide chain with three functional domains, namely the C-terminal domain, which is responsible for binding the toxin to the host cell membrane, the hydrophobic middle domain, which is made (co)responsible for the translocation process through the cellular membranes, and the N-terminal domain, which has a glycosyltransferase function and imparts the toxic activity of the molecule.
The uptake process of the toxins in the host cell is in fact not yet fully explained, however it is considered a fact that the toxins, after binding to a host cell receptor, arrive into the host cell by endocytosis, and that for the development of their toxicity the N-terminal catalytic domain is split off and is conveyed into the cytosol of the host cell. There, the catalytic domain glycolizes specifically GTPases of the Rho sub-family (Rho, Rac and Cdc42), which in turn are involved in an abundance of signal transduction cascades, and in this way blocks the respective signal transduction processes, which finally leads to the disaggregation of the cytoskeleton and to cell death.
In the prior art, hitherto it was assumed that the splitting off of the catalytic N-terminal domain of the toxin peptide chains of TcdA and TcdB and also other “large clostridial toxins” LCT being catalyzed by a cellular protease (Rupnik et al., 2005 and Pfeiffer et al., 2003). Corresponding evidence was not, however, able to be provided.
In the course of the investigations which form the basis of the present invention, it was now surprisingly found, however, that the cleavage of TcdA and TcdB is an autocatalytic process, which is initiated by inositol phosphate (IP), and that consequently the toxins of Clostridium difficile, in addition to their catalytic function of glycosyltransferase also have the function of a protease for auto-cleavage or autocatalytic cleavage.
This protease function was identified as aspartate protease. As catalytic centre of the protease function, the protein region was identified which comprises the amino acid sequence of amino acid position AS 1653 to AS 1678 of TcdB according to sequence No. P18177 (SwissProt/TrEMBL). The motif DXG (Rao et al. 1998) characteristic for aspartate proteases lies at the amino acid position 1665.
As inositol phosphate binding site, the protein region was identified which comprises the amino acid sequence of amino acid position AS 1517 to AS 2142 of the TcdB protein according to sequence No. P18177 (SwissProt/TrEMBL). This amino acid sequence constitutes an inosin-5-monophosphate-dehydrogenase (IMPDH) motif, which is composed of two regions, namely AS 1517-AS 1593 and AS 1918-AS 2142, which are separated by a 325 amino acid long protein section without sequence homology.
For the treatment of patients with C. difficile infections, firstly the initiating antibiotic is discontinued, in so far as this is possible. The further treatment takes place exclusively symptomatically. With a long-lasting or serious etiopathology, and when a discontinuance of the initiating antibiotic is not possible for other reasons, metrondiazol or vancomycin is administered for therapy.
The disadvantages of the current antimicrobial therapy are manifold. It is critical here above all that a disease which was initiated as a result of the treatment of a different infection situation with antibiotics can not be effectively healed with an antimicrobial therapy. The background to this is the fact that C. difficile only occurs relatively rarely in the gut of healthy people and can not stand up to the normal intestinal flora. If the normal intestinal flora is destroyed by antiobiotic therapy, C. difficile can establish itself and can effectively colonize the gut. The antibiotic therapy directed against C. difficile leads in turn to the destruction of the intestinal flora and thereby causatively also prevents the development of a healthy intestinal flora. This also explains the large number of remissions which are to be observed after completion of the antimicrobial therapy. An additional disadvantage of the current therapy is the increasing occurrence of multiresistant C. difficile strains in recent times. The threat thereby is that the sole therapy hitherto for diseases induced by C. difficile also will become useless and the number of deaths as a result of C. difficile diseases will increase. In addition to this is the fact that the antibiotics necessary for the treatment of a C. difficile infection are very expensive and normally are only used in justified cases as reserve antibiotics.